遗传表达的RNA比率型荧光生物传感及其活细胞成像新方法研究

Live cell imaging technology is an important research tool in life sciences and related disciplines. The development of quantitative fluorescence biosensing methods for live cell imaging is the trend of analysis chemistry. This project will focus on the key scientific issue of providing robust internal standards for ratiometric biosensing of live cells. For the purpose of quantitative fluorescence analysis of living cells, on the basis of light-up RNA aptamers and plasmid expression technologies, we will explore the feasibility of genetically co-expressing RNA aptamer and its robust fluorescence internal standard using a single promoter. Systematical study will be conducted on the general principles and biological basis of the design of single promoter-controlled plasmid for developing general methods for the construction of robust fluorescence internal standards based on genetic expression. Further, taking the detection of mRNA and ERK2 as examples, we will explore the signal condition manners for light-up RNA biosensing in live cells and the basic issues related to the application of live cell imaging. Based on the researches, we will sum up the basic mechanism and methods for developing genetically encoded RNA biosensors for ratiometric imaging in living cells. We are expecting to provide an effective technology platform for quantitative analysis of living cells in related biomedical, clinical diagnostic, and drug screening studies.

活细胞成像分析技术是生命科学及相关学科领域重要的研究工具。发展可定量的活细胞荧光生物传感方法是活细胞成像分析的发展趋势。本项目拟围绕比率型活细胞生物传感方法的稳健内标及其高效构建的关键科学问题，以活细胞定量荧光成像分析为目的，基于点亮型RNA适体及质粒表达技术，探讨基于单启动子转录事件控制RNA适体及其稳健荧光内标同步表达的合理性与可行性，系统研究单启动子质粒设计的通用原则及生物基础，发展基于遗传表达这一高效、简便方式构建稳健荧光内标的通用方法；更进一步，以mRNA与蛋白酶ERK2的检测为例，探索活细胞内点亮型RNA生物传感的信号调控方式及其活细胞分析应用相关的基础性问题，据此建立基于遗传表达的RNA比率型荧光生物传感的新原理与新方法，以期为相关生物医学、临床诊断、药物筛选等研究的活细胞定量分析提供有效的技术平台。

围绕比率型活细胞荧光生物传感的稳健内标及其高效构建的关键科学问题，以哺乳动物活细胞准确定量成像分析为目的，重点探索了基于单启动子转录事件控制RNA适体及其稳健荧光内标同步表达的合理性与可行性，以及单启动子质粒设计的通用原则及生物基础，发展了高效便捷的基于遗传表达的稳健荧光内标构建通用方法；同时，探索了活细胞内点亮型RNA生物传感的信号调控方式，并深入探讨了RNA适体折叠效率和稳定性等活细胞应用相关的基础性问题，据此建立了基于遗传表达的活细胞比率型荧光生物传感的新原理与新方法，有望为相关生物医学等领域研究的活细胞定量分析提供有效的技术平台。项目执行期间，诸多研究工作发表在同领域一流学术刊物上，得到国际同行的较高评价。相关研究成果已经形成SCI收录高水平学术论文12篇，均标注本项目资助，其中IF>5论文9篇，包括Chemical Science (2), ACS Nano (1), Analytical Chemistry (2), Chemical Communications (3)等生物传感器与相关领域重要国际学术刊物的论文。申请国家发明专利3件，已授权1件。同时，项目组联合培养博士后1名，博/硕士生11名，本科生3人，圆满完成了既定目标和任务。